Engineering a heavy atom derivative for the X-ray structure analysis of cyclodextrin glycosyltransferase |
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Authors: | Klein, Claudio Vogel, Wolfgang Bender, Hans Schulz, Georg E. |
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Affiliation: | Insutut für Organische Chemie und Biochemie der Universität Albertstr. 21, D-7800 Freiburg i.Br, FRG |
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Abstract: | Based on a preliminary structural model of cyclodextrin glycosyltransferasefrom Bacillus circulans (EC 2.4.1.19[EC]), Ser428 and Ser475 ofthe enzyme were mutated to cysteines in order to produce suitableheavy atom derivatives. Mutant Ser475 - Cys could not be expressedas protein. Mutant Ser428 - Cys was expressed in Escherichiacoli and purified. It crystallized isomorphously and gave riseto a mercury derivative that improved the electron density map.The structural results show that the new mercury-binding siteis in a pocket at the protein surface. |
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Keywords: | cyclodextrin glycosyltransferase/ heavy atom derivative/ mutant crystallization/ site-directed mutagenesis/ X-ray structure |
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